Hu et al. BMC Medical Genetics(2020) 21:213
https://doi.org/10.1186/s12881-020-01152-5

RESEARCH ARTICLE

Open Access

Novel variants of ABCA4 in Han Chinesefamilies with Stargardt disease
Fang-Yuan Hu1,2,3, Feng-Juan Gao1,2,3, Jian-kang Li4,5, Ping Xu1,2,3, Dan-Dan Wang1,2,3, Sheng-Hai Zhang1,2,3 andJi-Hong Wu1,2,3*

Abstract
Background: Stargardt disease (STGD1) is a common recessive hereditary macular dystrophy in early adulthood orchildhood, with an estimated prevalence of 1:8000 to 1:10,000. ABCA4 is the causative gene for STGD1. The current
study aims at identifying the novel disease-related ABCA4 variants in Han Chinese families with STGD1 using nextgeneration sequencing (NGS).
Methods: In the present study, 12 unrelated Han Chinese families (19 males and 17 females) with STGD1 were
tested by panel-based NGS. In order to capture the coding exons and the untranslated regions (UTRs) plus 30 bp of
intronic flanking sequences of 792 genes, which were closely associated with usual ophthalmic genetic disease, we
designed a customized panel, namely, Target_Eye_792_V2 chip. STGD1 patients were clinically diagnosed byexperienced ophthalmologists. All the detected variants were filtered and analyzed through the public databases
and in silico programs to assess potential pathogenicity.
Results: Twenty-one ABCA4 mutant variants were detected in 12 unrelated Han Chinese families with STGD1,
containing 14 missense, three splicing, two frameshift, one small deletion, and one nonsense variants. Base on theAmerican College of Medical Genetics (ACMG) guidelines, 8 likely pathogenic and 13 pathogenic variants were
determined. The functional consequences of these mutant variants were predicted through in silico programs. Of
the 21 mutant variants in ABCA4, two novel coding variants c.3017G > A and c.5167 T > C and one novel null variant
c.3051-1G > A were detected in three unrelated probands.
Conclusions: By panel-based NGS, 21 ABCA4 variants were confirmed in 12 unrelated Han Chinese families. Amongthem, 3 novel mutant variants were found, which further expanded the ABCA4 mutation spectrum in STGD1
patients.
Keywords: ABCA4 gene, STGD1, Han Chinese patients, Panel-based NGS, Novel mutant variants

Background
ABCA4, mapping to chromosome 1p22.1, encodes the
ATP-binding cassette transporter 4, which is a transmembrane protein highly expressed in the cone and rod
photoreceptors [1, 2]. This transmembrane protein acts
* Correspondence: jihongwu@fudan.edu.cn
1
Eye Institute and Department of Ophthalmology, Eye & ENT Hospital, Fudan
University, Shanghai, China
2
NHC Key Laboratory of Myopia (Fudan University); Key Laboratory of
Myopia, Chinese Academy of Medical Sciences, Shanghai, China
Full list of author information is available at the end of the article

as a flippase and mainly regulates the transmembranetransport of N-retinylidene-phosphatidylethanolamine
across the disc membranes in the outer segments ofcone and rod. The dysfunction of the ABCA4 protein results in the massive accumulation of A2E, a by-productof the visual cycle, in the retinal pigment epithelium(RPE) as lipofuscin deposits, which induces RPE apoptosis and secondary photoreceptor degeneration [3–6].
The mutant variants in ABCA4 are mainly responsible
for autosomal recessive retinal dystrophies, including© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate ifchanges were made. The images or other third party material in this article are included in the article's Creative Commonslicence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
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The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to thedata made available in this article, unless otherwise stated in a credit line to the data.

Hu et al. BMC Medical Genetics(2020) 21:213

Stargardt disease (STGD1; OMIM 248200) [1, 7], retinitis pigmentosa (RP19; OMIM 601718) [8, 9], cone-rod
dystrophy (CRD3; OMIM 604116) [10, 11], and earlyonset severe retinal dystrophy (OMIM 248200) [12, 13].
According to the Human Gene Mutation Database(HGMD), more than 1200 disease-associated ABCA4
variants are already found in retinopathy, including missense, nonsense, splicing, frameshift, small insertion ordeletion, and gross insertion or deletion variants. Amongthem, approximately 80% of variants in ABCA4 are associated with STGD1.
STGD1 is an inherited macular atrophic disease inchildhood or early adulthood, following an autosomal recessive inheritance pattern [12, 14]. It is typically characterized by central visual impairment and macularatrophy [14–17]. The underlying gene for STGD1 is
ABCA4 [1]. In our previous study, we have completed
ABCA4 gene screening among a large Chinese population with STGD1 to extend the ABCA4 mutation
spectrum and identified three prevalent ABCA4 variants,
namely, c.101_106delCTTTAT, c.2894A > G, and c.6563
T > C [18]. Although many ABCA4 variants have beenreported, we found that novel ABCA4 mutant variants
were still found in STGD1 patients.
In the present study, we analyzed the mutation
spectrum of ABCA4 in 12 Han Chinese families with
STGD1 by next-generation sequencing (NGS). Our analysis revealed 21 ABCA4 mutant variants and the functional impacts of these variants were evaluated by insilico programs. Among them, 3 novel mutant variants
were determined, namely, two missense and one splicing
variants.

Methods

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the current study, all subjects underwent corresponding
genetic testing by panel-based NGS. The genomic DNA
of all subjects was extracted from peripheral blood by
QIAGEN FlexiGene DNA Kit (Qiagen, Venlo, theNetherlands) according to the manufacturer’s protocols.
Then, the genomic DNA was sonicated to shear intofragments, including the promoter and flanking intronic
regions and the coding exons. The enriched libraries
were sequenced by BGISEQ-500 [19].
Genetic analyses and variant identification

Sequencing reads that passed quality control werealigned to the reference sequences (UCSC hg 38)
through the Burrows-Wheeler Aligner (BWA) program [20]. The reference sequence number for
ABCA4 is NM_000350. We analyzed the minor allele
frequencies (MAFs) of all identified mutant variantsin the four databases, namely, ExAC (http://exac.
broadinstitute.org), dbSNP (http://www.ncbi.nlm.nih.
gov/projects/SNP/), ESP6500 (http://evs.gs.washington.
edu/EVS/), and 1000 Genomes Project (http://browser.1000genomes.org/). The variants with MAFs>
0.1% were filtered out to eliminate benign variants.
After filtering, the mutant variants were further
screened according to variant report, the potential
deleterious effects, and genotype-phenotype correlation in the three major databases, including OMIM
(http://www.omim.org/), HGMD (http://www.hgmd.cf.
ac.uk/ac/index.php), and ClinVar (https://www.ncbi.
nlm.nih.gov/clinvar/) [21, 22]. Variants were determined as likely pathogenic or pathogenic based on
the American College of Medical Genetics (ACMG)
guidelines. Candidate variants were validated bySanger sequencing.

Subjects

From June 2019 to October 2019, 36 subjects from 12
unrelated Han Chinese families with STGD1 were recruited at Fudan University Eye Ear Nose and Throat
Hospital. Ophthalmologic examination was performedon the subjects, containing spectral domain optical coherence tomography (SD-OCT), electroretinography(ERG), fundus autofluorescence (FAF), slit-lamp biomicroscopy, fundus photograph, and best corrected visualacuity (BCVA). In addition, the family and medical histories were recorded. STGD1 patients were clinicallydiagnosed by experienced ophthalmologists.
Library preparation and panel-based NGS

We cooperated with BGI-Shenzhen to design a speciallycustomized capture panel, named as Target_Eye_792_V2
chip, which could capture the exons and the untranslated regions (UTRs) plus 30 bp of intronic flanking
sequences of 792 genes closely associated with usual
ophthalmic genetic disease (Supplementary Table S1). In

Results
Clinical findings

Twelve unrelated Chinese families (19 males and 17
females) with STGD1 underwent genetic testing by
panel-based NGS in this study. The basic clinical phenotypic characteristics of 12 probands were shown in
Table 1. The vast majority of probands showed early onset age, mainly in early childhood or adolescence.
Among them, the longest disease duration was morethan 20 years. The visual acuity of probands was significantly decreased in both eyes, and some patients complained of an increased difficulty in adapting to darkness
and abnormal color vision. In addition, all parents
showed the normal phenotypes, and the pedigrees of 12
families were obtained (Fig. 1). Fundus examination inprobands revealed the atrophy of the bilateral RPE
around the macula. Representative photographs of patient F1:II:2 were shown in Fig. 2. Fundus photographs
showed some pigment mottling and yellow-white flecks(2020) 21:213

Hu et al. BMC Medical Genetics

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Table 1 The ABCA4 disease-associated variants identified in this study
ProbandID Age
Sex(years)
F1:II:2

F2:II:1

F3:II:1

F4:II:1

F5:II:1

F6:II:1

19

31

8

22

39

28

Age of onset BCVA
Exon/
(years)(OD/OS) Intron

Female 10

Male

10

Female 2

Male

15

Female 34

Female 23

NucleotidechangeAmino acid change Variant
type

Zygosity References

0.15/0.15 Exon22

c.3287C > Tp.(Ser1096Leu)

Missense

Het

Stone et al.
(2017)

Exon12c.1561delG

p.(Val521Serfs*47)

Frameshift

Het

Wang et al.
(2018)

0.05/0.02 Exon22

c.3262C > Ap.(Pro1088Thr)

Missense

Het

Hu et al.
(2019) [18]0.05/0.1

Exon2

c.101_
p.(Ser34_Leu35del)106delCTTTATInframeDeletion

Het

Huang et al.
(2018)

Exon27c.4066C > Tp.(Gln1356*)

Nonsense

Het

Hu et al.
(2019) [18]Exon22c.3262C > Ap.(Pro1088Thr)

Missense

Het

Hu et al.
(2019) [18]p.[Thr1821Aspfs*6,
Thr1821Valfs*13]

Splicing

Het

Sangermano
et al.
(2018)

0.06/0.06 Intron39 c.5584 + 5G >A
Exon8c.1019A > Gp.(Tyr340Cys)

Missense

Het

Nassisi et al.
(2018)

0.15/0.05 Exon42c.5882G > Ap.(Gly1961Glu)

Missense

Het

Wiszniewski
et al.
(2005)

Intron13 c.1761-2A > G p.?

Splicing

Het

Jiang et al.
(2016) [23]Exon14c.2034G > Tp.(Lys678Asn)

Missense

Het

Zaneveld
et al.
(2015)

Exon14c.2063_2064insAp.(Asn688Lysfs*78)

Frameshift

Het

Jiang et al.
(2016) [23]c.2894A > G

p.(Asn965Ser)

Missense

Het

Zernant et al.
(2011)

Intron21 c.3051-1G > A p.?

Splicing

Het

This study

Exon19c.2894A > G

p.(Asn965Ser)

Missense

Het

Zernant et al.
(2011)

Exon38c.5327C > Tp.(Pro1776Leu)

Missense

Het

Mandal et al.
(2005)

Exon12c.1760G > Ap.(Arg587Lys)

Missense

Het

Fujinami
et al.
(2015) [14]Exon4c.428C > Tp.(Pro143Leu)

Missense

Het

Consugar
et al.
(2015) [21]0.2/0.15 Exon13c.1892G > Tp.(Gly631Val)

Missense

Het

Hu et al.
(2019) [18]Exon9c.1229 T > C

p.(Ile410Thr)

Missense

Het

Jiang et al.
(2016) [23]Exon22c.3322C > Tp.(Arg1108Cys)

Missense

Het

Sodi et al.
(2006)

Exon20c.3017G > Ap.(Gly1006Asp)

Missense

Het

This study

Exon36

c.5167 T > Cp.(Tyr1723His)

Missense

Het

This study

Exon2c.101_
p.(Ser34_Leu35del)106delCTTTAT

0.3/0.2

F7:II:1

28

Male

3

0.07/0.05 Exon19

F8:II:1

22

Male

7

0.01/0.1

F9:II:1

F10:II:2

10

20

Female 8Male10

0.1/0.5

F11:II:2

13

Male

11

0.3/0.6

F12:II:1

16

Male

14

0.05/0.1

Abbreviations: BCVA best corrected visual acuity, OD right eye, OS left eye. [*] represents termination codon

Inframe Deletion Het

Huang et al.
(2018)

Hu et al. BMC Medical Genetics(2020) 21:213

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Fig. 1 Pedigrees of 12 Han Chinese families and identified mutants. Circles and squares indicate females and males, respectively. The darkened
shapes indicate the affected members and the empty shapes indicate the subjects with normal phenotype. “N” represents a wild type allele and“V” represents a mutant allele

in bilateral macula and a beaten-bronze macular appearance (Fig. 2a). Fundus autofluorescence (FAF) displayed
the fluorescence blocking due to macular pigment mottling and the multiple hyper-fluorescent dots corresponding with subretinal flecks detected in fundus
photographs (Fig. 2b). Macular OCT revealed macularatrophy, reduced thickness of the retinal outer layers,
and the alteration of choroidal reflectivity (Fig. 2c).
Detected variants and genetic analyses

Twenty-one disease-related ABCA4 variants wereidentified in 12 Han Chinese families, containing 18
known and 3 novel mutant variants (Fig. 1, Table 1).
As presented in Supplementary Table S2, thedetailed genetic analyses and the functional evaluation of 21 distinct variants were summarized. Twomutant variants were identified in all probands andall of them were identified as compound heterozygotes. Except for the parent F8: I: 1 who was notdetected, all parents of the proband were confirmed
to possess one ABCA4 variant and clinicallydiagnosed as normal phenotype. In addition, theelder brother of the proband F1:II:2 also possessed
one heterozygous variant, while the other siblings
with the normal phenotype in families 9, 10, and 11
did not receive genetic testing.
The detected ABCA4 variants contained 14 missense,
three splicing, two frameshift, one small deletion, andone nonsense variants. The pathogenicity of the variants
was determined based on the ACMG guidelines, including 8 likely pathogenic and 13 pathogenic variants. In
our previous studies, we have confirmed three prevalent
ABCA4 variants in STGD1 patients from China [18].
Among them, two frequent variants, c.101_106delCTTTAT and c.2894A > G, were detected in four families in
the present study. In families 2 and 12, variants
c.3262C > A and c.5167 T > C were found in trans withc.101_106delCTTTAT, respectively. Variants c.30511G > A and c.5327C > T were found in a compound
heterozygous state with c.2894A > G in families 7 and 8,
respectively. Furthermore, c.3262C > A, c.4066C > T, andc.1892G > T, which were first identified by us in STGD1

Hu et al. BMC Medical Genetics(2020) 21:213

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Fig. 2 The representative images of ophthalmologic examination from proband F1:II:2. a Fundus photographs exhibiting macular atrophy andyellow-white spots in the posterior pole. b FAF exhibiting macular hypo-autofluorescence corresponding with atrophy and multiple hyperfluorescent dots corresponding with subretinal flecks. c OCT exhibiting the significant thinning of retinal outer layers in macular areaHu et al. BMC Medical Genetics(2020) 21:213

patients [18], were also found in three families, namely,families 2, 3, and 10.
Of the 21 mutant variants, 18 detected variants were
distributed in 12 exons of the ABCA4 gene. And in
introns 12, 21, and 39 of ABCA4, three splicing variants
were identified. Based on the analyses of the Human
Splicing Finder (HSF), c.1761-2A > G in intron 12 andc.3051-1G > A in intron 21 mainly affected splice acceptor sites. Variant c.5584 + 5G > A in intron 39 could
affect splice donor sites (Supplementary Table S2).
Moreover, our analyses revealed that the identified variants
were mainly located in exocytoplasmic domains 1 (ECD1)
and nucleotide-binding domain 1 (NBD1) (Fig. 3a). The
NBD1 domain was regarded as one of the high incidence
regions of variants in STGD1 patients from Chinese population in our previous study [18].
Novel variants in the ABCA4 gene

Three novel ABCA4 mutant variants were identified
in three unrelated families, namely, one splicing variant and two missense variants (Table 1). Two novel
missense variants, c.3017G > A p.Gly1006Asp andc.5167 T > C p.Tyr1723His, were detected in families
11 and 12, respectively. In family 11, variantc.3017G > A, located in the NBD1 domain, was found

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in trans with c.3322C > T in proband F11:II:2. In proband F12:II:1, variant c.5167 T > C, located in transmembrane domain 2 (TMD2), was detected incompound heterozygosity with c.101_106delCTTTAT(Table 1, Fig. 3a). As presented in Fig. 3b, glycine
residue at position 1006 and tyrosine residue at position 1723 were highly conserved in the ABCA4 protein across different species, suggesting that these twovariants, p.Gly1006Asp and p.Tyr1723His, might result in a deleterious effect. In accordance with the
ACMG guidelines, two novel heterozygous variants
were categorized as likely pathogenic variants. Moreover, one novel spicing variant c.3051-1G > A wasfound in proband F7:II:1 and detected in trans withc.2894A > G. On the basis of the HSF analyses,
c.3051-1G > A was predicted to affect the normal splicing. Variant c.3051-1G > A was identified as likely
pathogenic in accordance with the ACMG guidelines.

Discussion
In the current study, 21 ABCA4 mutant variants were
found in 12 unrelated Han Chinese families with STGD1
by genetic testing. Among them, three novel variants
were identified.

Fig. 3 Distribution of the detected mutants in the ABCA4 functional domains and conservation analyses. a Location of 21 mutants in six
functional domains. Black: missense mutant. Red: splicing mutant. Purple: frameshift mutant. Green: nonsense mutant. Yellow: small deletionmutant. b Multiple orthologous sequence alignment displaying the conserved amino acid residues (Glycine 1006 and Tyrosine 1723)

Hu et al. BMC Medical Genetics(2020) 21:213

Except F5: II: 1, the onset age of all probands with
STGD1 was relatively early. The mean onset age in 12
families was 12.25 years (range 2–34 years), which wasyounger than that of the previously reported patientswith STGD1. The average age of onset in these reported
cohorts is mostly around 20 years [18, 24, 25]. However,some studies have also shown that the onset age of
STGD1 patients is earlier, with the average age of onset
between 10 and 14 years [23, 26, 27].
In our previous research, c.101_106delCTTTAT p.Ser34_
Leu35del, c.6563 T > C p.Phe2188Ser, and c.2894A > G
p.Asn965Ser have been confirmed to be three prevalent
ABCA4 variants in STGD1 patients mainly from eastern
China [18]. Interestingly, two frequent variants c.101_
106delCTTTAT and c.2894A > G were also detected in 4
of 12 unrelated families in the present study. Based on thefact that all of the 12 families also come from eastern
China, such results further illustrate the high frequency of
the two mutant variants in this region. Our analyses haverevealed that the allele frequencies of these three prevalentvariants are about 20% [18], which are lower than those ofsome high frequency variants in STGD1 patients from Europe. For instance, the frequencies of p.Gly1961Glu andp.[Leu541Pro;Ala1038Val] may exceed 30% in European
populations [25, 28]. Meanwhile, in another study of
ABCA4 gene screening for STGD1 patients in Chinese
population, the frequencies of the prevalent variants(namely, c.101_106delCTTTAT, c.2424C > G, c.2894A > G,and c.6563 T > C) are actually only 14% [23]. According to
the above findings, we speculated that the prevalence of
STGD1 in China might be lower than that in European
populations. So far, there is no exact statistical data on the
STGD1 prevalence in Chinese populations. It is necessary
to conduct the epidemiological investigation on a larger
number of patients in the future, so as to better understandthe disease characteristics of STGD1 in Chinese patients.
In addition, the common mutant variant c.5882G > A
p.Gly1961Glu identified in eastern Africa and Europe[28, 29] was also detected in family 5, and c.1761-2A > G
was detected in trans with c.5882G > A in proband F5:II:
1. It has been reported that STGD1 cases harboring thep.Gly1961Glu allele tend to have a mild disease phenotype, and patients with variant p.Gly1961Glu in either
the homozygous or heterozygous states show delayed
onset of symptoms and later age of onset. Moreover,p.Gly1961Glu is closely related to the bull’s eye maculopathy phenotype of STGD1 [30–32]. Consistent withprevious researches, the proband F5:II:1 carrying
p.Gly1961Glu presented the highest age of onset (34years) among all patients and the FAF imaging showed
typical bull’s eye maculopathy.
Furthermore, variants c.3262C > A, c.4066C > T, andc.1892G > T that were first confirmed by our group [18]were also detected in three unrelated families in thisPage 7 of 10

study. In proband F10:II:2, c.1892G > T was found incompound heterozygosity with c.1229 T > C and bothvariants were located in the ECD1 domain. Variant
c.3262C > A was detected in two families. In particular,c.3262C > A and c.4066C > T were detected in proband
F3:II:1 who was identified as compound heterozygotes.
These results further indicated the potential deleteriousness caused by the three ABCA4 mutants.
Of the 21 disease-associated ABCA4 mutants detected in the current study, 18 known variants weredetected in all probands, including 12 missense, two
splicing, two frameshift, one small deletion, and one
nonsense mutants. Among them, 16 known mutants
were distributed in 12 exons of the ABCA4 gene. Analysis revealed three dissimilar mutants in exon 22; twodissimilar mutants each in exons 12 and 14; and one
mutant each in exons 2, 4, 8, 9, 13, 19, 27, 38, and 42.
In addition, two known splicing mutants were identified in two introns of ABCA4 (introns 12 and 39). As
presented in Supplementary Table S2, the function
prediction of coding variants was performed by four
software prediction programs, including MutationTaster, SIFT, FATHMM, and LRT. Through the analyses
with four online tools, the functional deleteriousnessof 12 known missense mutants was verified by at least
three of the four prediction programs. Three severe/null variants, namely, one nonsense mutant c.4066C >T and two frameshift mutants c.1561delG and c.2063_
2064insA, could introduce a premature truncatingcodon in the protein during translation. The three severe/null variants and one inframe deletion variantc.101_106delCTTTAT were predicted to have deleterious effects through the MutationTaster analyses.
Moreover, two known splicing variants, c.5584 + 5G >A and c.1761-2A > G, mainly affect the splice donorsite and the splice acceptor site, respectively. The detected known variants of ABCA4 were distributed in
all protein functional domains except for the exocytoplasmic domain 1 (ECD1). On basis of the online tools
analyses and ABCA4 protein structure, p.Lys678Asn
and p.Pro1776Leu located in the TMD1 and TMD2
domains might disrupt the transmembrane alphahelices and give rise to the dysfunction of ABCA4 protein transport. Variants p.Pro143Leu, p.Tyr340Cys,
p.Ile410Thr, p.Arg587Lys, and p.Gly631Val in the
ECD1 domain might have an impact on the topologically associated domains outside the cell and lead tothe loss of protein function. Variants p.Gly1961Glu,
p.Arg1108Cys, p.Ser1096Leu, p.Pro1088Thr, andp.Asn965Ser in the NBD1 and NBD2 domains could
affect the ATP hydrolysis function of protein throughdamaging the topologically associated domains incells. The functional impacts of p.Asn965Ser havebeen demonstrated in the corresponding in vivo andHu et al. BMC Medical Genetics(2020) 21:213

in vitro studies [33, 34]. The pathogenicity of thesevariants was determined based on the ACMG guidelines, containing 5 likely pathogenic and 13 pathogenic
mutants.
Three novel ABCA4 variants were found in this study,
including one splicing and two missense variants. All
three mutants showed the dramatically low allelic frequency in dbSNP, 1000 Genomes Project, ExAC, and
ESP6500 databases and were not observed in 200 normal
controls. Two novel missense mutants, c.3017G > A
p.Gly1006Asp and c.5167 T > C p.Tyr1723His, cosegregated with the clinical phenotype of STGD1 in families 11 and 12. In silico analyses of missense variants revealed that c.3017G > A and c.5167 T > C could cause
the functional damage in all of four software prediction
programs (MutationTaster, LRT, SIFT, and FATHMM).
Meanwhile, all of these two variants were detected intrans with the known pathogenic variants (c.3322C >T and c.101_106delCTTTAT), further indicating their
possible pathogenicity. More significantly, c.3017G > A
was located in a high incidence region of variants,
namely, the NBD1 domain [18, 23]. Moreover, codons
1006 and 1723 of ABCA4 were strictly conserved
amino acids among dissimilar species, suggesting thatthese amino acids were essential for the normal protein function. According to the ABCA4 protein structure and the online tools analyses, p.Gly1006Asp in
the NBD1 domain might destroy the topologically associated domains in cells, thus interfering with theATPase activity of the protein. And p.Tyr1723His in
the TMD2 domain might bring about the ABCA4transport dysfunction through breaking the extracellular topological domains. A novel null mutant, c.30511G > A in intron 21, was identified and co-segregatedwith the clinical phenotype of disease in family 7. As
shown in Fig. 1, c.3051-1G > A was found in trans withc.2894A > G, which has been confirmed to be a high frequency variant in Chinese populations [18, 23]. According
to HSF analyses, variant c.3051-1G > A affects the splice acceptor site. The above-mentioned analysis results
confirmed the potential pathogenicity of these three novel
mutants. In accordance with the ACMG guidelines, threenovel mutants were categorized as likely pathogenic
variants.
In the process of screening patients with the clinicalphenotype of STGD1, we also detected the variants ofother pathogenic genes instead of ABCA4 related tomacular degeneration in another three Chinese families,
namely, GUCY2D (c.2513G > A), PDE6C (c.967 T > C
and c.1579C > T), and POC1B (c.1153G > A andc.458C > T). All of them are the causative genes for coneor cone-rod dystrophy [35–37]. The phenotypes ofmacular degeneration caused by these three genes arevery similar to STGD1. The results also fully

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demonstrate the importance of genetic testing in clinicaldifferential diagnosis.

Conclusions
In conclusion, 21 disease-related ABCA4 variants wereidentified in 12 unrelated Han Chinese families with
STGD1, and the functional influences of the detectedmutants were assessed by the software prediction programs. Furthermore, two novel missense and one novel
splicing variants were found in three unrelated probands,
extending the mutation spectrum of ABCA4 in patientsfrom Chinese population. The detailed genetic
characterization of STGD1 patients will contribute to
the clinical differential diagnosis of macular degeneration and provide reliable information for genetic
counselling.
Supplementary Information
The online version contains supplementary material available at https://doi.
org/10.1186/s12881-020-01152-5.
Additional file 1: Table S1. Gene list of capture panel.
Additional file 2: Table S2. Twenty-one ABCA4 variants identified inthis study and their in silico functional assessment.
Abbreviations
AMD: Age-related macular degeneration\; ACMG: American College ofMedical Genetics; BCVA: Best corrected visual acuity; BWA: Burrows-Wheeler
Aligner; CRD: Cone-rod dystrophy; ERG: Electroretinography;
ECD: Exocytoplasmic domain; FAF: Fundus autofluorescence; HGMD: Human
Gene Mutation Database; HSF: Human Splicing Finder; MAFs: Minor allele
frequencies; NGS: Next-generation sequencing; NBD: Nucleotide-binding
domain; RPE: Retinal pigment epithelium; RP: Retinitis pigmentosa;
STGD1: Stargardt disease; SD-OCT: Spectral domain optical coherence
tomography; TMD: Transmembrane domain; UTRs: Untranslated regions
Acknowledgments
We sincerely thank all of the patients and families who agreed to participate
in this study.
Authors’ contributions
JW and FH designed and conceived this study. JW, FH, FG, PX, and DW
recruited patients, and performed clinical examinations and interpretation.
FH, FG, DW, and SZ collected the clinical samples and clinical data. JW, FH,
FG, and JL analyzed the sequencing data. JW and FH wrote and revised themanuscript. All authors read and approved the final manuscript.
Funding
This work was supported by the National Natural Science Foundation ofChina (Grant NSFC81770925, NSFC81790641), Xuhui District Health andFamily Planning Commission Key Disease Joint Project (XHLHGG201807), theNon-profit Central Research Institute Fund of Chinese Academy of Medical
Sciences (2018PT32019), Outstanding Academic Leaders in Shanghai(20XD1401100), Program for Outstanding Medical Academic Leader(2019LJ01), and Aging and women’s and children’s health Special project ofShanghai Municipal Health Commission (2020YJZX0102). The funding body
had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.
Availability of data and materials
Our data generated and/or analysed during the current study is available in
the ABCA4 LOVD (www.lovd.nl/ABCA4), which is an almost complete variant
and case registry for ABCA4. Here is the link: https://databases.lovd.nl/shared/
individuals?search_owned_by_=%3D%22Fangyuan%20Hu%22

Hu et al. BMC Medical Genetics(2020) 21:213

Ethics approval and consent to participate
This study was performed in accordance with the Code of Ethics of theWorld Medical Association (Declaration of Helsinki) for medical research
involving human subjects and was approved by the Ethics Committee ofFudan University Eye Ear Nose and Throat Hospital. Written informed
consent was obtained from all individuals who participated in this study andfrom parents on behalf of minors.
Consent for publication
Written informed consent for publication of identifying images or otherpersonal or clinical details was obtained from all of the participants or theparents or legal guardians of any participant under the age of 18.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Eye Institute and Department of Ophthalmology, Eye & ENT Hospital, Fudan
University, Shanghai, China. 2NHC Key Laboratory of Myopia (Fudan
University); Key Laboratory of Myopia, Chinese Academy of Medical Sciences,
Shanghai, China. 3Shanghai Key Laboratory of Visual Impairment andRestoration, Shanghai, China. 4BGI-Shenzhen, Shenzhen, China. 5Departmentof Computer Science, City University of Hong Kong, Kowloon, Hong Kong.
Received: 24 March 2020 Accepted: 19 October 2020

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